Project Summary The long-term goal of our work is to understand the molecular mechanisms through which G protein-coupled receptors (GPCRs) are activated and attenuated. These receptors are the largest family of membrane proteins in the human genome, and a major target for pharmaceutical drugs. We primarily study rhodopsin and its affiliate proteins. Although structural details about these proteins are now well known, the dynamic events involved in their interactions and regulation are not. The driving force behind the current proposal is the fact that we still do not know how the retinal ligands that modulate rhodopsin activity enter and leave the receptor, or why interactions of rhodopsin with its signaling partners transducin and arrestin affect these events. Aim 1 will test our new hypothesis that rhodopsin behaves like a traditional ligand binding GPCR, with its ability to bind different ligands being governed by conformational selection. Specifically, we will pursue our exciting discovery that under some conditions, the agonist, all-trans retinal (ATR), binds to the receptor in equilibrium. We will also determine if this potential retinal equilibrium binding is altered in several disease causing rhodopsin mutations. Aim 2 focuses on using and improving novel rhodopsin-based fluorescent biosensors we have recently developed. Our ultimate goal is to use these sensors to address new research questions and to screen small molecule libraries to identify novel, non-retinal ligands that bind rhodopsin either orthosterically or allosterically and act as agonists or antagonists. We feel these goals are important for vision research ? the stability of the retinal linkage varies widely among different opsins and is a factor in some visual disease states. Aberrations in the ability of the eye to deal with the large fluxes of retinal under bright light conditions is thought to be a major contributing factor to diseases like atrophic age-related macular degeneration (AMD), one of the leading causes of vision loss in people over 60. Thus, a better understanding of the processes underlying retinal binding and release is fundamentally important. We also think identifying new compounds that modulate rhodopsin activity will have significant impact. Although rhodopsin was the first GPCR ever identified, there are still no small molecule drugs that bind rhodopsin with high affinity and modify its behavior. If our hypothesis is correct and retinal binding and release sometimes occurs in dynamic equilibrium, it may be possible to treat rhodopsin as a ?druggable? GPCR, and identify compounds that can modulate its activity. We think that realizing this goal is long overdue and thus our proposal is very appropriate for the R21 funding mechanism.